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Detection of Herpes Simplex Virus DNA in Semen of Men With Genital HSV-2 Infection

Wald, Anna MD, MPH*†; Matson, Pam BS; Ryncarz, Alexander PhD‡§; Corey, Lawrence MD*‡§

Sexually Transmitted Diseases: January 1999 - Volume 26 - Issue 1 - p 1–3
Original Articles

Background and Objectives: Previous studies, using viral culture, have suggested that herpes simplex virus (HSV) isolation from semen is rare. This study attempts to investigate further the role of semen in sexual transmission of HSV.

Goals of this Study: To evaluate semen samples for HSV DNA with a sensitive polymerase chain reaction (PCR) test.

Study Design: Laboratory examination of 255 stored semen samples collected from 15 healthy men with genital HSV-2 infection during a prospective clinical trial.

Results: Herpes simplex virus DNA was detected in 8 (3.1%) semen samples, 6 of which were collected during a herpes recurrence. Herpes simplex virus DNA was not detected in any of the 18 samples collected during acyclovir therapy.

Conclusion: Herpes simplex DNA can be detected in semen, although it appears closely associated with clinical HSV reactivation. More detailed studies will be needed to assess the role HSV-2 in semen plays in transmission of infection.

From the Departments of Medicine*, Epidemiology†, and Laboratory Medicine‡, University of Washington, and Program in Infectious Diseases§, Fred Hutchinson Cancer Research Center, Seattle, Washington

Supported by Herpes Program Project Grant NIH AI-30731.

Correspondence and reprint requests: Anna Wald, MD, MPH, University of Washington, Virology Research Clinic, 1001 Broadway, Suite 320, Seattle, WA 98122.

Received for publication June 15, 1998, revised September 21, 1998, and accepted September 28, 1998.

INFECTION WITH HSV-2 affects 17% of men and 26% of women in the United States.1 As with many other sexually transmitted infections, the efficiency of transmission of herpes simplex virus (HSV) appears higher from men to women than women to men.2 For infections characterized by urethral discharge, ejaculate is thought to serve as a vehicle for transmission of the pathogen. For genital herpes, the differences in prevalence rates may include differences in the genital epithelium in men and women and the higher rate of recurrences in men than women.3

In a previous study of semen from men with recurrent genital herpes, HSV was not isolated in culture from 827 semen samples.4 However, acquisition of HSV-2 resulting from artificial insemination with sperm obtained from a man with asymptomatic primary HSV-2 infection has been documented.5 This suggests that HSV can actively replicate in the male genitourinary tract, although the exact sites of reactivation are unknown. To investigate the presence of HSV in semen samples and its potential role in HSV transmission, we used polymerase chain reaction (PCR) assay on semen specimens, which were negative for virus by culture.

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Methods

The patient population and study design have been described previously.4 Briefly, men with culture-confirmed HSV-2 infection were enrolled in a study designed to evaluate the effect of daily acyclovir on sperm production. Patients were followed for 13 months: a 4-month observation period, a 6-month treatment period, and a 3-month follow-up period without antiviral therapy. During treatment, subjects were randomized 1:1:1 to acyclovir 200 mg twice a day, acyclovir 200 mg five times a day, or placebo. The subjects provided semen samples twice a week throughout the study. For this study, 255 semen samples from 15 men were retrieved which had been stored frozen at −20°C. The odds ratio (OR) for detection of HSV DNA during a time of a recurrence versus not during a time of a recurrence was estimated with logistic regression for grouped data.

The methods for the PCR assay have been described.6,7 Briefly, DNA was extracted from 0.2 ml of whole semen using 0.4 ml of 4M guanidinium thiocyanate followed by two rounds of 1:1 phenol: chloroform/isoamyl (24:1). DNA was precipitated using one volume of isopropanol, 0.067 volume of 3M sodium acetate, and 20 μg of glycogen, then placed at −20°C for 30 minutes, centrifuged at 14,000 xg for 30 minutes, and aspirated, leaving the glycogen/DNA pellet undisturbed. The DNA was then washed with ethanol and taken up in 50 μl of 10 mM Tris. HSV DNA was amplified using a specific primer set. The upper primer HSV gB (1978-1997) sequence is 5′-CTG GTC AGC TTT CGG TAC GA. The lower primer HSV gB (2319-2300) sequence is 5′-CAG GTC GTG CAG CTG GTT GC. DNA was amplified for 35 cycles: denaturing at 96°C, annealing at 54°C, and extending at 72°C. A homogeneous internal control was added to the PCR mixture and amplified simultaneously using the above mentioned primers to eliminate false negative results.7 Polymerase chain reaction products were analyzed using a liquid hybridization technique.7 An HSV specific probe, 2A probe with sequence AGG TCG ATG AAG GTG CTG ACG GTG GTG A, was labeled with 32-P and added to the PCR product. A probe specific for the internal control sequence was mixed with the same amount of product in a separate tube. The hybridization mixtures were denatured at 97°C for 5 minutes, allowed to cool to 25°C in 15 minutes, and then run on a 2% agarose gel. The gels were dried and autoradiography was performed using X-Omat film at room temperature overnight.

To determine the sensitivity of the HSV DNA PCR assay for detection of HSV DNA in semen, we performed an experiment in which a known amount of HSV-2 was added to semen from a HSV seronegative person, then diluted serially to establish the lower level of detection. We found that our assay consistently detected as few as five copies of HSV DNA/0.2 ml of semen sample.

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Results

Two hundred fifty five semen samples from 15 men were analyzed. All samples were negative for HSV by viral culture. Herpes simplex virus DNA was detected in semen from 7 (47%) men and in 8 of 255 (3.1%) semen samples.

We reviewed the clinical information about the participants to determine whether the participants were receiving antiviral therapy at the time the samples were collected and whether the samples were collected during a recurrence of genital herpes. Two hundred thirty seven samples were obtained during an observation period when the participants were not receiving daily acyclovir and 21 samples were obtained during acyclovir administration (Table 1). Among the semen samples obtained without antiviral therapy, 6 of 53 (11.3%) specimens collected during a genital herpes recurrence had HSV DNA detected compared with 2 of 184 (1.1%) specimens collected during asymptomatic periods, OR = 13.3 (95% CI, 1.7, 101). Of interest, HSV DNA was not detected in any of 18 semen samples collected during acyclovir therapy.

TABLE 1

TABLE 1

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Discussion

Our study indicates that HSV-2 DNA can be found in semen. Of interest, HSV DNA was detected in 11% of semen samples collected during a clinical recurrence of genital herpes and in 1% of samples obtained on days without lesions. This rate of HSV DNA detection in semen is lower than the subclinical shedding rate in the genital and perianal skin of heterosexual men that we have previously studied and similar to that of urethral specimens.8

Prior studies of semen obtained from men with genital herpes have failed to yield HSV in tissue culture.4,9,10 Men participating in these studies had a history of recurrent genital herpes, thus a virologically and immunologically established infection. The role of HSV in the semen in the transmission of HSV is unclear, although HSV DNA can on occasion be detected in semen on days without lesions. However, there was a higher frequency of HSV detection during recurrences than during nonlesional days.

Our PCR assay has been shown to detect viral shedding 2.5 times more frequently than viral isolation in specimens of genital secretions.11 Although the amount of HSV critical for transmission of infection has not been established, HSV culture-negative, PCR-positive episodes of viral shedding have been documented to result in HSV transmission from women to neonates at delivery.12 Furthermore, bovine herpesvirus type 1 (BHV-1), a significant problem in animal husbandry, has been shown to cause infection during artificial insemination via transmission in semen, even when viral cultures fail to yield infectious virus.13 As such, PCR has been established as a standard assay for screening bovine semen prior to insemination.14,15 However, although BHV-1, also an alphaherpesvirus, appears to share many characteristics of human herpes simplex viruses, BHV-1 shedding in semen is common and can often be detected by viral isolation.16

Our study cannot identify the origin of the virus found in semen. Although the PCR assay is quite specific, one area we cannot control is the potential contamination of the semen sample during collection. It is possible that contact with genital lesions during masturbation could have resulted in some of the positive results during recurrences. Although asymptomatic shedding from the urethra is rare,17 HSV can be isolated from the penile skin on about 2% of days without lesions in immunocompetent men with genital herpes.8 Samples from penile skin swabs for PCR assay were no longer available from this cohort of men, and, as such, a more detailed study in which multiple skin sites and semen samples are obtained, especially among clinically asymptomatic men, is required. However, because we found that the HSV DNA was present in the semen on fewer days than it would be expected to be shed asymptomatically from the penile skin, contact with semen is likely to be less important than contact with lesions on genital skin.

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References

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